The invention relates to a method of obtaining cells of osteoblast phenotype from cells present in human extramedullary adipose tissue. The resulting cells can be used for making bone implants.
It is known that in order to remedy losses of bone tissue following injury or surgical operations, it is possible to implant replacement or filler materials. These materials can be bone grafts or artificial products such as porous ceramics, or else natural products such as coral skeleton.
The making of allografts comprises in particular risks of transmitting certain serious viral diseases. The making of autografts is more satisfactory from this point of view, but taking the graft requires surgical intervention, which presents considerable risks of morbidity.
For these reasons, it has been recommended to use implants based on biocompatible, and possibly biodegradable materials, such as tricalcium phosphate, hydroxyapatite, plaster, coral, polymers based on poly(lactic acid), etc. Macroporous materials are particularly advantageous since the presence of pores enhances bone regrowth.
For several years, research has been directed towards using cells having osteogenic potential, optionally in combination with biomaterials; see for example French patent No. 2 679 250 which contemplates culturing osteoblasts on a porous three-dimensional solid support constituted by coral skeleton for the purpose of being implanted in patients suffering from a loss of bone substance, that has already occurred or that is expected (when surgical resection is planned).
The interest of such methods naturally lies in enabling implants to be made using autologous cells.
In this field, research has been directed towards using bone marrow cells which are capable of differentiating into osteoblasts, in particular under the influence of certain growth factors that have an osteoinducing effect. It is indeed known that bone marrow cell cultures are capable of tending in particular towards development of osteoblast phenotypes.
However, taking bone marrow presents the same drawbacks as taking bone grafts.
Studies performed on the rabbit have shown that cells of the stroma-vascular compartment of extramedullary adipose tissue are capable of differentiating into osteoblasts in in vitro cultures in the presence of the BMP2 osteo-inducing factor and dexamethasone; see L. Lecoeur et al., Cellular Engineering, Vol. 2, No. 2, 1-7 (1997). Dexamethasone on its own does not make this differentiation possible.
It has now been discovered that, on the contrary, in vitro culturing of certain human extramedullary adipose tissue cells can lead to osteoblast differentiation in the presence of a glucocorticoid on its own, e.g. in the presence of dexamethasone on its own, i.e. not associated with an osteo-inducing factor. The use of such factors, which are expensive products, is not necessary.
Of the various osteo-inducing factors, mention can be made of the protein mediator known as xe2x80x9cbone morphogenetic proteinxe2x80x9d (BMP), described by M.R. Urist et al., P.N.A.S. USA 76: 1828-1832 (1979). That terminology covers in fact various osteo-inducing protein factors (from BMP2 to BMP9), see for example Yamaguchi et al., Sem. Cell. Biol. 6, 165-173 (1993).
The invention thus provides a method for inducing osteoblast differentiation and/or for obtaining cells engaged in said differentiation, starting from human extramedullary adipose tissue cells, the method comprising the step consisting in incubating said starting cells in a liquid nutrient medium for a period of time sufficient to enable said cells to develop, said nutrient medium containing a solution of at least one glucocorticoid and being free from adipogenic factor, and in particular free from insulin.
In other words, the method of the invention is a method in which starting cells are cultivated and in which at least one glucocorticoid is added to the culture medium in order to induce osteoblast differentiation and/or to obtain cells engaged in said differentiation.
H. Haunder et al. in J. Clin. Invest. 84: 1663-1670 (1998) have described differentiation of human subcutaneous adipose tissue cells into adipocytes by culturing in the presence of insulin and of glucocorticoids.